Metabolic Resistance And Regulation Mechanism Of Nitenpyram In Nilaparvata Lugens

The brown planthopper?BPH?,Nilaparvata lugens?St?l?,is a crucial pest of rice crops throughout Asian countries.However,due to the indiscriminate use of insecticides,N.lugens has developed varying degrees of resistance to many kinds of insecticides including nitenpyram.Previous studies have found that the enhanced activities of cytochrome P450 monooxygenase and esterase were related to nitenpyram resistance in N.lugens,but there was a lack of in-depth research on the metabolic resistance mechanism mediated by them.Based on this,in the present study,the crossresistance spectrum and the functions of important P450 and CarE genes involved in nitenpyram-resistant BPH were investigated.Moreover,miRNAs involved in nitenpyram resistance were identified by transcriptome sequencing technology,and the function of miRNA in the nitenpyram resistance of N.lugens was explored from the perspective of post-transcriptional regulation.The main research results are as follows: 1.Resistance selection and biochemistry mechanism of nitenpyram resistance in N.lugensTwo nitenpyram resistant strains?NR,RR=164.18-fold;XY,RR=203.35-fold?were evolved through successive selection for several generations from a laboratory susceptible strain?NS?and a field population?XY?collected from Xinyang,Henan,in 2017.The bioassay results showed that the NR exhibited cross-resistance to imidacloprid,thiamethoxam,clothianidin,dinotefuran,sulfoxaflor,cycloxaprid,etofenprox and isoprocarb but not to triflumezopyrim,chlorpyrifos and buprofezin.The synergists of piperonyl butoxide?PBO?and triphenyl phosphate?TPP?significantly increased nitenpyram toxicity in the two resistant strains,and the activities of P450 and CarE increased significantly in the NR and XY compared with that of the NS strain,it is speculated that P450 and CarE are involved in the evolution of the resistance of N.lugens to nitenpyram.2.Identification and functional study of P450 genes involved in nitenpyram resistance in N.lugensTranscriptome sequencing was performed on the resistant and susceptible strains of N.lugens through the Illumina sequencing platform.Compared with the susceptible strain,a total of 1561 genes were significantly upregulated and 986 genes were significantly downregulated in the resistant strain.The expression levels of fifty-four P450 genes in the susceptible strain NS and two resistant strains?NR and XY?by quantitative real-time PCR were performed.The results showed that a total of 9 P450 genes?CYP6ER1,CYP6FL3,CYP3115A1,CYP418A1,CYP302A1,CYP305A15,CYP4C76,CYP4FB1 and CYP439A1?were significantly up-regulated in two resistant strains.Among them,the expression levels of CYP6ER1,CYP302A1 and CYP3115A1 showed the same trend as the resistance levels of nitenpyram.Moreover,RNAi results showed that silencing CYP6ER1,CYP302A1 and CYP3115A1 can significantly restore the susceptibility of N.lugens to nitenpyram,and transgenic expression of CYP6ER1 in Drosophila melanogaster significantly reduced the susceptibility to nitenpyram,indicating that overexpression of CYP6ER1 mediated nitenpyram resistance in N.lugens.3.Identification and functional study of CarE genes involved in nitenpyram resistance in N.lugensA total of 29 CarE genes were identified based on genome data and transcriptome sequencing data in N.lugens.Phylogenetic tree analysis showed that the 29 CarE genes were divided into seven clades in which the ?-esterase clade was significantly expanded.Tissue-specific expression analysis indicated that 15 ?-esterase genes were abundantly distributed in the midgut or fat body,and two acetylcholine esterase genes were highly expressed in the head.The expression of sixteen CarE genes was significantly induced in response to the challenge of nitenpyram.RT-q PCR revealed that CarE1,CarE2,CarE9 and CarE19 were significantly upregulated in the two resistant strains,and the RNAi further clarified that overexpression of CarE1 mediated nitenpyram resistance in N.lugens.4.Identification and regulation mechanism of miRNA involved in nitenpyram resistance in N.lugensSilencing Dicer,AGO and Drosha with RNAi significantly upregulated the expression levels of CYP6ER1,CYP302A1,CYP3115A1 and CarE1,suggesting that miRNA is involved in the metabolic resistance of N.lugens to nitenpyram.Using Small RNA sequencing,a total of 221 miRNAs were identified in N.lugens,of which 179 were novel miRNAs.Furthermore,compared with the susceptible strain,a total of 36 miRNAs were significantly downregulated and 36 miRNAs were significantly upregulated in the resistant strain.In addition,novel₈5 and novel₁91 were predicted to target CYP6ER1 and CarE1 via miRNA-target gene prediction software.Dual luciferase reporter assays further confirmed that CYP6ER1 and CarE1 is modulated by novel₈5 and novel₁91 through binding to the ORF region,respectively.The suppression of CYP6ER1,CYP302A1,and CYP3115A1 through injection of ds RNA led to significant susceptibility in two resistant strains.The expression of CYP6ER1 and CarE1 is inhibited when the novel₈5 and novel₁91 mimics were injected respectively,resulting in significantly increased susceptibility of N.lugens to nitenpyram.In contrast,the injection of novel₈5 and novel₁91 inhibitors significantly increased the resistance of N.lugens to nitenpyram.These results indicate that novel₈5 and novel₁91 could be involved in nitenpyram resistance by regulating the expression of CYP6ER1 and CarE1,respectively.These findings provide an important theoretical basis for comprehensively revealing the metabolic regulation mechanism of N.lugens to nitenpyram.